Long-term storage, transport and archiving of nucleic acids on filter paper or chemically modified matrices is a well-known technique for preserving genetic material before the DNA or RNA is extracted and isolated in a form for use in genetic analysis such as PCR. Thus, EP 1563091 (Smith et al, Whatman) relates to methods for storing nucleic acids from samples such as cells or cell lysates. The nucleic acid is isolated and stored for extended periods of time, at room temperature and humidity, on a wide variety of filters and other types of solid support or solid phase media. Moreover, the document describes methods for storing nucleic acid-containing samples on a wide range of solid support matrices in tubes, columns, or multiwell plates.
WO 90/03959 (Burgoyne) describes a cellulose-based solid support for the storage of DNA, including blood DNA, comprising a solid matrix having a compound or composition which protects against degradation of DNA incorporated into or absorbed on the matrix. This document also discloses methods for storage of DNA using the solid medium, and for recovery of or in situ use of DNA.
U.S. Pat. No. 5,705,345 (Lundin et al.) describes a method of nucleic acid preparation whereby the sample containing cells is lysed to release nucleic acid and the sample is treated with cyclodextrin to neutralize the extractant. The advantage of this system is that conventional detergent removal requires a separation step however with the addition of cyclodextrin to neutralize the detergent it would remove the separation step needed and reduce chance of contamination.
U.S. Pat. No. 5,496,562 (Burgoyne) describes a cellulose-based solid medium and method for DNA storage. Method for storage and transport of DNA on the solid medium, as well as methods which involve either (a) the recovery of the DNA from the solid medium or (b) the use of the DNA in situ on the solid medium (for example, DNA sequence amplification by PCR) are disclosed. The method described incorporates a surfactant or detergent on the surface of the solid medium and would require a separate step for the removal of the detergent before PCR is performed as a detergent would interfere with the PCR process.
EP 2290099 B1 (Qiagen) describes again a method for processing and amplifying DNA. The method includes the steps of contacting the sample containing DNA to a solid support wherein a lysis reagent is bound to the solid support. The DNA is subsequently treated with a DNA purifying reagent and is purified.
WO96/39813 (Burgoyne) describes a solid medium for storing a sample of genetic material and subsequent analysis; the solid medium comprising a protein denaturing agent and a chelating agent. The method described is for chelating agents which are any compound capable of complexing multivalent ions including Group II and Group III multivalent metal ions and transition metal ions.
WO 96/18731 (Deggerdal) describes a method of isolating nucleic acid whereby the sample is bound to a solid support and the sample is contacted with a detergent and subsequent steps performed to isolate the nucleic acid.
WO 00/53807 (Smith, Whatman) describes a medium for the storage and lysis of samples containing genetic material which can be eluted and analyzed. The medium is coated with a lysis reagent. In addition the medium could be coated with a weak base, a chelating agent, a surfactant and optionally uric acid.
WO 99/38962 (Health, Gentra Systems Inc.) describes a solid support with a bound lysis reagent. The lysis reagent can comprise of a detergent, a chelating agent, water and optionally an RNA digesting enzyme. The solid support again would require further steps for purification of the nucleic acid for amplification analysis.
Inhibitors of PCR
There are significant problems with inhibitory substances or inhibitors interfering with polymerase chain reactions on nucleic acids, including nucleic acids stored or immobilized on solid supports such as cellulose-derived filters. Sources of inhibitors can be the materials and reagents such as detergents that come into contact with samples during processing or nucleic acid purification. These include excess KCl, NaCl and other salts, ionic detergents such as sodium deoxycholate, sarkosyl and sodium dodecyl sulphate (SDS), ethanol and isopropanol, phenol (see Burgoyne WO 90/03959) and others. All users of PCR are likely to be impacted by inhibitors at some time, but the wide range of forensic sample types and variety of sampling conditions encountered make forensic scientists particularly vulnerable.
The presence of chemicals that interfere with PCR analysis in samples is well-known and a number of approaches are documented that describe their removal. However, none of these approaches are particularly efficient. In particular the application of samples to solid supports, particularly cellulose-derived materials, increases the problems with PCR inhibition because of the inclusion of potent chemicals such as SDS and chaotropic salts which are typically used to stabilize nucleic acids on the solid support (see Burgoyne WO 90/03959).
PCR inhibitors usually affect PCR through interaction with DNA or interference with the DNA polymerase. In a multiplex PCR reaction, it is possible for the different sequences to experience different inhibition effects to varying degrees, leading to a disparity in results. Alternatively, various inhibitors reduce the availability of cofactors (such as magnesium) or otherwise interfere with the interaction of PCR cofactors with the DNA polymerase. Some of the pitfalls of quantitative real-time reverse transcription polymerase chain reaction, including the effect of inhibitors, are described by Bustin & Nolan (J. Biomolecular Techniques, 2004, 15, 155-166).
Current methods for inhibitor removal are often carried out during the DNA purification procedure by binding to single or double stranded DNA covalently linked to a support. However, this is a tedious process and prior art methods have a number of clear disadvantages in terms of cost, complexity and in particular, user time.
U.S. Pat. No. 5,705,345 (Lundin et al.) describes a method of nucleic acid preparation whereby the sample containing cells is lysed to release nucleic acid and the sample is treated with cyclodextrin to neutralize the extractant. The advantage of this system is that conventional detergent removal requires a separation step however with the addition of sequestrants such as cyclodextrin to neutralize the detergent it would remove the separation step needed and reduce chance of contamination.
WO 2010/066908 (Beckers et al.,) describes the use of cyclodextrins to improve the specificity, sensitivity and/or yield of PCR. The method claimed is an amplification reaction which is performed in a reaction mixture comprising at least one cyclodextrin and performing the amplification reaction on said reaction.
Alternative methods involve the binding of nucleic acids in the presence of chaotropic agents such that DNA binds to silica or glass particles or glass beads. This property was used to purify nucleic acid using glass powder or silica beads under alkaline conditions. Typical chaotropic agents include guanidinium thiocyanate or guanidinium hydrochloride and recently glass beads have been substituted with glass containing minicolumns. While the chaotropic agent may neutralize the interference of the detergent on the PCR process, the chaotropic agent itself may reduce the efficiency of PCR amplification.
Wang et al. (Enzyme Microbiology Technology, 1990) describes a method of recovering protein from a bacteria by exposing E. coli to certain combinations of guanidine and Triton X-100 in a solution. It was noted that neither of the chemicals alone and at low concentrations extracts more than 10% of intracellular protein however together were able to release over 50% overall protein.
U.S. Pat. No. 5,939,259 (Schleicher & Schuell Inc.) describes a method for the collection and storage of nucleic acids by coating an absorbent material with a solution containing from 0.5M to 2.0M chaotropic salt and allowed to dry. Other ingredients can be added to the invention in order to enhance lysis or disruption of intact cells, bacteria and viruses which include anionic, cationic or zwitterionic surfactants, such as Tween 20 or Triton X-100.
Given the wide range of PCR inhibitor-laden sample types and the options available for handling them, a multifaceted approach is the best solution for amplification failure. There is a need to reduce any potential PCR inhibitors used in the nucleic acid collection process wherein the nucleic acid is immobilized on a solid support and thereby improve PCR amplification.
The present invention addresses this problem and provides methods and kits which can be used for the collection of nucleic acids using solid supports, particularly cellulose-derived supports.